The invention relates to a device and method for programmably controlling electrophoresis performed by use of either invariant or variable (pulsed) fields. More specifically, the present invention is directed toward a programmable control of the direction and magnitude of the electrical field experienced by a particle in a rotatable gel bed. The temperature of the gel bed and sample particles placed therein is also programmably regulated through a thermoelectric chilling device such as a Peltier device. The invention utilizes a user-programmable control board for multiplexing the control of both electric field (magnitude and direction) and temperature during either pulsed field gel (PFG) or invariant field gel electrophoresis. By programming the various conditions that determine the electrophoretic fractionation, the present invention increases the significance and usefulness of electrophoretic analysis of various complex particles such as proteins, peptides, or nucleic acids.
By far the most common method of analyzing particles, such as DNA molecules, is electrophoresis. Molecules or particles having net electrical charges may be analyzed by observing their migration in a gel subjected to the application of electrical fields. The migrations are dependent upon many factors which include but are not limited to the nature of electric fields, the pH of solvents containing the particles, the temperature of the solvent and the characteristics of the particles themselves. By changing the direction and magnitude of the electric field during electrophoresis, the length of DNA molecules which are separable has been increased to between 3.0-6.0 Mb. Thus, changing the direction of electrical field impinging upon the particles and/or the pulse duration and magnitude of the electric field will produce increased separation of either large DNA molecules or, presumably, other particles forming either a random coil or a flexible rod. Changing the direction of the impinging electrical field is typically achieved by either changing the electrical potential on vertical electrodes or rotating the gel in which migratory DNA-type particles are placed. Contained both over and within the gel is an electrically conductive solvent which conducts electrical current that, in the case of a rotating gel, is driven by an electrical potential from electrodes horizontally displaced adjacent the rotating bed. As the bed and particles rotate about a fixed axis, the electrical field angle of incidence upon the particles changes.
Although changing the magnitude and direction of electric fields improves fractionation of larger linear or open-circular DNA particles, there still remains a limit as to the size of particles that can be analyzed. For linear DNA molecules having lengths that exceed 3.0-6.0 Mb, additional procedures are needed. Further, when DNA particles have branches or other types of irregular protrusions (such as proteins, for example), improved control of electrical potential gradient (direction and magnitude) and temperature is needed to fractionate the particle. Oftentimes, when fractionating large particles, it is necessary to increase electrical potential gradients by injecting more power into the electrophoretic device. Increased power causes heat build-up within and across the gel bed thereby adversely affecting electrophoresis readings. A fast and efficient way of dissipating heat away from the particles and gel bed is needed when migrating large particles using higher power input levels.
Not only are conventional devices unable to analyze large linear (&gt;6 Mb) or open-circular (&gt;0.3 Mb) DNA particles, but they also suffer from poor adaptability in analyzing different forms of particles. Each time a different DNA particle is sought for analysis, the apparatus must be changed or retrofitted to accommodate the particular size, shape or composition of that particle. For example, a smaller particle may respond poorly to discontinuous or continuous rotation of the gel bed during electrophoresis. In such a case, it may be desirable to provide unidirectional electrophoresis without changing or retrofitting the electrophoretic apparatus. Still further, it may even be desirable to provide more accurate readings when, for example, performing a highly resonant separation, i.e., one that has a steep dependance on parameters of electrophoresis. In such a case, uniformity and control of both electrical potential gradient and temperature is critical.
Perhaps the greatest difficulty when performing electrophoresis is to perform repetitive tests using the same electric field gradient and temperature for each test. Thus, a need arises for programmably and accurately controlling, preferably with programmable storage and recall, electrophoretic conditions. Furthermore, the electrophoretic device must have the capacity for accurate control during successive tests, such that the conditions remain constant for each test. Current devices are not known for their ability to control temperature and electrical potential; most do not store and recall control parameters for repetitive electrophoresis operations. Further, current devices do not multiplex control of two or more electrophoretic disks or electrophoretic devices, nor can current devices programmably control multiple modes of variation of the electrical field and other control parameters values, for example, a mode can be the temporal sequence of disk rotations and electrical source (magnitude and polarity).